Journal of Antimicrobial Chemotherapy (2005) 55, 347 351 doi:10.1093/jac/dki013 Advance Access publication 4 February 2005 Concentrations and in vivo antibacterial activity of spiramycin and metronidazole in patients with periodontitis treated with high-dose metronidazole and the spiramycin/ metronidazole combination Pierre-Pascal Poulet 1, Danielle Duffaut 1, Pierre Barthet 1 and Ivan Brumpt 2 * JAC 1 Laboratory of Oral Biology, School of Dental Medicine, Toulouse; 2 Laboratoire Aventis, Paris, France Received 18 June 2004; returned 6 August 2004; revised 1 December 2004; accepted 8 December 2004 Objectives: Previous studies have shown that metronidazole, alone or in combination with spiramycin (250 mg/1 500 000 units, three times/day), is an effective treatment for active periodontitis, although the dose of metronidazole currently used (750 mg/day) could provide concentrations in gingival crevice fluid that are too low for the MICs of the involved pathogens. This study tested the in vivo antibacterial efficacy of the currently used metronidazole dose (as contained in the fixed spiramycin/metronidazole combination) in patients with an active periodontitis, and of a high dose (1500 mg/day) of metronidazole alone. Methods: We measured the MICs of spiramycin and metronidazole for the recovered pathogens and the gingival crevice fluid antibiotic concentrations of both antibiotics, and attempted to correlate them with bacterial eradication. Results: The concentrations of metronidazole consistently exceeded the MICs for the pathogens isolated in the corresponding sites, even at the usual metronidazole (250 mg three times/day) dose. All the bacterial species were eradicated during treatment and at follow-up, although Fusobacterium spp. eradicated during treatment reappeared in a majority of the cases at follow-up, 30 days after treatment, in both groups. Conclusions: The results of antibiotic therapy with metronidazole or the spiramycin/metronidazole combination are consistent with their in vitro antibacterial activity and with the local antibiotic concentrations; they suggest that the currently used metronidazole dose (250 mg, three times/day) alone or as part of the spiramycin/metronidazole combination, could be sufficient for the treatment of active periodontitis. Keywords: oral anaerobic bacteria, gingival crevice fluid, pharmacodynamics Introduction The bacterial flora of periodontitis is complex and encompasses obligate anaerobes (Gram-negative bacilli and Gram-positive cocci), fastidious anaerobes and capnophilic bacteria. Amoxicillin (combined or not with clavulanic acid), metronidazole (750 mg/day), or combinations of amoxicillin and metronidazole, or metronidazole and ciprofloxacin, have been proposed. 1 The spiramycin/metronidazole combination is widely used in France and Canada, on the basis of the complementary antibacterial spectrum of both agents, the possibility of in vitro and in vivo synergy on Bacteroides spp. and the high concentrations of spiramycin in tissues. 2 Previous studies have shown that metronidazole in combination with spiramycin (250 mg/1 500 000 units, three times/day), is an effective treatment of active periodontitis. 3 Metronidazole alone has also been shown to be effective, although the dose used (750 mg/day) could provide gingival crevice fluid concentrations that are too low for the MICs of some pathogens, such as Micromonas micros or actinomycetemcomitans. 4 6 The objective of this study was to test the in vivo antibacterial efficacy of the currently used metronidazole dose (as contained in the fixed spiramycin/metronidazole combination) in patients with active periodontitis, and of a high dose (1500 mg/day) of... *Corresponding author. Tel: +33-1-55-71-05-58; Fax: +33-1-55-71-05-10; E-mail: ivan.brumpt@aventis.com... 347 JAC vol.55 no.3 q The British Society for Antimicrobial Chemotherapy 2005; all rights reserved.
P.-P. Poulet et al. metronidazole alone, to measure the gingival crevice fluid concentrations, and to attempt to correlate the bacterial eradication results with the antibacterial activity of these agents in vitro and the concentrations measured in the gingival crevice fluid. Patients and methods This was a prospective, parallel-group, open, randomized, multicentre, pilot study. Eighteen patients with periodontitis in an active phase were included; 10 patients received the spiramycin/metronidazole combination (1 500 000 units/250 mg, three times a day), and eight patients received high-dose metronidazole alone (500 mg three times a day), both for 6 days. The study medications were provided by Laboratoire Aventis, Paris, France. A bacterial sample was taken for culture at three dental sites (two affected and one nearby spared site) at inclusion, and the sampling was repeated in the same sites at day 6 (on-treatment) and at day 30 (follow-up). The sample was collected on sterile paper points, inserted gently in the subgingival sulcus after cleaning of the subgingival plaque and avoiding any contamination by saliva. It was left in situ for 15 s, and placed in a sealed vial containing a specific transport medium (VGAM III), and transported within 4 h to a central microbiology laboratory (Department of Oral Biology, School of Dental Medicine, Toulouse, France) where the sample was processed immediately. Selective and non-selective media were used, incubated in anaerobic conditions for culture of strict anaerobes (Gram-negative bacilli and Grampositive cocci) and in a 5% CO 2 enriched atmosphere for culture of capnophilic Gram-negative bacilli. The following pathogens were identified by biochemical methods (criteria of the Virginia Polytechnic Institute and the Pasteur Institute) and classified according to the combination pathogenic complexes described by Socransky: Porphyromonas gingivalis, Tannerella forsythia (Bacteroides forsythus), Treponema denticola (red complex), Fusobacterium spp., Prevotella intermedia, M. micros (orange complex), associated with the subgingival plaque; and A. actinomycetemcomitans, Capnocytophaga spp., Eikenella corrodens (green complex) and Campylobacter rectus (mauve complex), associated with the supragingival plaque. 7 The susceptibility to metronidazole and spiramycin was tested by dilution in agar according to the M11A3 standard of the NCCLS, up to the 8 mg/l susceptibility breakpoint for metronidazole. 8 The MIC of spiramycin was tested up to 32 mg/l. In addition, the MIC of metronidazole was determined after the addition of a fixed dose of spiramycin as well as the MIC of spiramycin after the addition of a fixed dose of metronidazole. The Fusobacterium spp. strains, cultivated before treatment and at follow-up in the same patient, were not subjected to genotyping analysis. The crevice fluid was collected by adsorption on a sterile paper tip inserted as above, and according to the previous description by Rotzetter et al. 5 A sample from the same sites was taken for the concentration assay of metronidazole and spiramycin on days 2, 6 (3 h after the morning dose) and 7 (24 h after the last dose), and frozen at 80 8C until the end of the study. The measurements of both compounds in the gingival crevice fluid were made using a specific liquid chromatography method with simultaneous detection by mass spectrometry (LC MS/MS) developed by CEPHAC, Saint- Benoît, France, with a 150 mg/l quantification limit. The imprecision and inaccuracy of the method were, for metronidazole, < 7% and ±3%, respectively; for spiramycin I, the imprecision and inaccuracy were < 9% and ±6%, respectively (inter-assay variation). The range of concentrations for the controls was 0.5 40 mg/l. The protocol was approved by the local Ethics Committee and the patients gave informed consent to participation in the study prior to enrolment. Results In vivo antibacterial activity The bacterial eradication on day 6 and day 30, according to the species found at enrolment, are presented in Table 1. All bacterial species were eradicated on day 6 in the spiramycin/ metronidazole group, except for A. actinomycetemcomitans and one strain of Capnocytophaga spp., but Fusobacterium spp. colonized a large number of sites on day 30. All bacterial species were eradicated on day 6 in the metronidazole group, except for Capnocytophaga spp., and E. corrodens. P. intermedia was Table 1. Bacterial species isolated at enrolment, and during and after treatment (number of sites involved by each species) Spiramycin/metronidazole Metronidazole affected site (n = 20) spared site (n = 10) affected site (n = 16) spared site (n =8) Bacterial species D0 D6 D30 D0 D6 D30 D0 D6 D30 D0 D6 D30 Porphyromonas gingivalis 3 3 Tannerella forsythia 5 Fusobacterium spp. 7 13 4 3 6 9 1 2 Prevotella intermedia 11 2 1 1 7 3 7 1 Micromonas micros 2 1 1 2 1 5 3 ND 2 ND 1 ND ND actinomycetemcomitans Capnocytophaga spp. 11 1 ND 4 ND 12 8 ND 2 1 ND Eikenella corrodens 1 ND ND 4 2 ND ND Campylobacter rectus 6 ND ND 3 ND 1 ND Number of sites with at least one of the above 19 4 14 7 5 15 8 9 3 1 3 ND, pathogen not evaluated at D30;, no pathogen of the species cultivated at the date. 348
Pharmacodynamics of metronidazole in periodontitis often present on day 6 and, like Fusobacterium spp. colonized a large number of sites on day 30. MIC in vitro The distribution of the MICs of metronidazole and spiramycin for each bacterial species isolated at enrolment is presented in Tables 2 and 3. P. gingivalis and P. intermedia appeared susceptible to both agents, whereas Fusobacterium spp. was susceptible to metronidazole, but not to spiramycin. M. micros was susceptible to metronidazole, but not to spiramycin. A. actinomycetemcomitans and E. corrodens are resistant to both agents. Capnocytophaga spp. was resistant to metronidazole, but also frequently to spiramycin for which a wide distribution of MICs of this agent was observed. C. rectus strains appeared susceptible to metronidazole, but not to spiramycin. The addition of a fixed dose of spiramycin to metronidazole, and of a fixed dose of metronidazole to spiramycin, did not result in a change in the MICs of metronidazole and spiramycin, respectively. Concentration in the gingival crevice fluid In the spiramycin/metronidazole group, the mean, median and extreme (minimum/maximum) concentrations of metronidazole in the gingival crevice fluid (affected and spared sites together) were 36.7 and 22.5 mg/l (min/max: 2.5/148 mg/l) at D2, 44.7 and 21.5 mg/l (min/max: 6.2/144 mg/l) at D6 and 43.9 and 3.4 mg/l (min/max: 0/350 mg/l) at D7. In the high-dose metronidazole group, they were 72.0 and 41.5 mg/l (min/max: 3.6/260 mg/l) at D2, 51.8 and 19.7 mg/l (min/max: 1.95/253 mg/l) at D6, 31.9 and 10.4 mg/l (min/max: 3.75/130 mg/l) at D7. In the spiramycin/metronidazole group, the mean, median and extreme (minimum/maximum) concentrations of spiramycin were 5.5 and 2.8 mg/l (min/max: 0/68.4 mg/l) at D2, 3.6 and 3.2 mg/l (min/max: 0/10.2 mg/l) at D6, and 4.3 and 3.2 mg/l (min/max: 0/26.9 mg/l) at D7. The concentrations were below the quantification limit in one patient (in the spared site only) at all dates (D2, D6 and D7), and in another patient (in an affected site) at D6 and D7. Inhibitory ratio For metronidazole, the MICs for A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens frequently exceeded the upper value tested (8 mg/l), but were not precisely measured. In consequence, when the in situ concentration of metronidazole in the corresponding sites exceeded 8 mg/l, the inhibitory ratio could not be calculated. In the remaining cases, the inhibitory ratio of metronidazole was constantly superior or equal to 1 for all pathogens in both groups, except for two strains of Capnocytophaga spp. in both groups. The inhibitory ratio of spiramycin (spiramycin/metronidazole group) was superior to 1 only for Table 2. Distribution of the MICs of metronidazole at enrolment MIC (mg/l) Bacterial species <_ 0.06 0.12 0.25 0.50 1 2 4 8 >8 Porphyromonas gingivalis (n =6) 2 2 1 1 Fusobacterium spp. (n = 15) 1 1 8 2 3 Prevotella intermedia (n =18) 1 1 6 7 1 1 1 Micromonas micros (n =4) 1 2 1 8 actinomycetemcomitans (n =8) Capnocytophaga spp. (n = 29) 2 1 3 3 20 Eikenella corrodens (n =5) 5 Campylobacter rectus (n =10) 1 3 2 1 3 Table 3. Distribution of the MICs of spiramycin at enrolment MIC (mg/l) Bacterial species <_ 0.06 0.12 0.25 0.50 1 2 4 8 16 32 >32 Porphyromonas gingivalis (n =6) 1 2 2 1 Fusobacterium spp. (n = 15) 1 1 5 2 4 2 Prevotella intermedia (n = 18) 8 1 6 1 1 1 Micromonas micros (n =4) 1 3 1 7 actinomycetemcomitans (n =8) Capnocytophaga spp. (n = 29) 1 1 7 9 7 1 2 1 Eikenella corrodens (n =5) 1 4 Campylobacter rectus (n = 10) 1 1 1 1 2 4 349
P.-P. Poulet et al. P. gingivalis and P. intermedia (2/2 and 10/10 strains, respectively); it was inferior to 1 for a large majority of Fusobacterium spp., A. actinomycetemcomitans, C. rectus and E. corrodens strains; it was superior to 1 in 7/14 Capnocytophaga spp. strains. Clinical efficacy and tolerance The clinical activity, evaluated by the evolution of the loss of attachment under treatment and at follow-up, showed an improvement or stabilization on day 6 in 16/20 affected sites in the spiramycin/metronidazole group and in 14/16 sites in the metronidazole group, and similarly on day 30 in 18/20 and 15/16 affected sites, respectively. Three patients in the high-dose metronidazole group presented a spontaneously resolving, mild or moderate adverse event (nausea, headache, cutaneous rash), whereas none was reported in the spiramycin/metronidazole group (P = 0.07). Discussion The study of the evolution of the bacterial flora showed that the spiramycin/metronidazole combination (1 500 000 U/250 mg) and high dose metronidazole (500 mg), given three times a day for 6 days, were effective, and presented little difference in efficacy between them. The contribution of spiramycin to the antibacterial efficacy of the spiramycin/metronidazole combination appears limited, except for Capnocytophaga spp., which appeared more frequently persistent on metronidazole alone, a finding consistent with the high MICs of metronidazole, whereas the MICs of spiramycin were lower. The clinical significance of this finding is, however, open to debate. On the other hand, Fusobacterium spp. colonized a large number of sites on day 30 in both groups, although metronidazole appeared active in vitro. In the absence of a comparison between the genotypes of the strains cultivated in the same patient before treatment and at follow-up, it is not possible to distinguish between relapse or re-infection. P. intermedia reappeared more frequently in the metronidazole group than in the spiramycin/metronidazole combination group, suggesting the possibility of an in vivo synergistic action between spiramycin and metronidazole. In this study, M. micros appeared susceptible to metronidazole (although the number of strains is limited) but presented a tendency to relapse on day 30. A. actinomycetemcomitans was resistant to both agents and appeared to be persistent on-treatment. The concentrations of metronidazole reached in the gingival crevice fluid of the patients in our study were particularly high, exceeding those measured by Rotzetter et al., 5 which were in the order of 10 mg/l after a single administration of 4 500 000 U/750 mg (spiramycin/metronidazole), or after a dose regimen of three daily administrations of 1 500 000 U/250 mg. No clear explanation can be offered for this discrepancy. In any case, the concentrations measured in our study largely exceeded the MICs of this agent for the involved pathogens, including with the usual dose regimen (250 mg unit doses). The results of the in vitro susceptibility tests of the above pathogens to spiramycin and metronidazole are generally consistent with the data already known for these agents by Poulet et al. 9 in patients with periodontal disease and Dubreuil et al. 10 in patients with infections from dental origin. Poulet et al. 9 and Dubreuil et al. 10 reported a low level of susceptibility to erythromycin of Fusobacterium spp., but good activity by metronidazole. To our knowledge, although a number of in vitro, descriptive bacteriological studies have been performed on the pathogens involved in periodontitis, and some clinical studies have described the in vivo, including long-term, bacteriological efficacy of different antibiotics, only few data are available on the pharmacodynamic rationale of the antibiotic treatment of this pathology. The concentrations of the agents in the gingival fluid have been described, but no correlation between the in vivo bacteriological efficacy and the antibiotic dose used, or the concentration reached in the gingival crevice fluid has yet been published. Our study provides an insight into the in vivo pharmacodynamic activity of antibacterial agents used in periodontitis, such as metronidazole or the spiramycin/metronidazole combination. In conclusion, the spiramycin/metronidazole combination (1 500 000 U/250 mg) and high dose metronidazole (500 mg), three times/day for 6 days, eliminate most of the bacterial pathogens on treatment and at follow-up (except for Fusobacterium spp.) with only slight differences between the groups. The concentrations of metronidazole are very high in the gingival crevice fluid and exceed the MICs for the involved pathogens, even at the usual metronidazole (250 mg three times/day) dose, and these results are consistent with the in vivo efficacy. These findings, together with the adverse events recorded in the high-dose metronidazole group, suggest that the use of metronidazole at the usual dose, alone or as part of the spiramycin/metronidazole combination, could be sufficient for the treatment of active periodontitis. Acknowledgements This work was supported by a financial grant from Laboratoire Aventis, France. Dr I. Brumpt is an employee of Laboratoire Aventis. References 1. 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